It once seemed as if bone growth was stopped after the bone was fully grown, but this is never true. In a body, bones always repeat formation and resorption (metabolism) and thereby a dynamic balance is maintained. At the cell level, osteoblasts which originate from osteoprogenitor cells play an important role in bone formation, and osteoclasts which originate from hematopoietic stem cells play an important role in bone resorption. A circle of bone formation stage.fwdarw.pause of bone formation.fwdarw.bone resorption stage.fwdarw.bone formation stage, is called remodeling.
The bone formation stage includes:
1st step: differentiation and proliferation of osteoblasts; PA0 2nd step: activation of osteoblasts; and PA0 3rd step: calcification of bone matrix. PA0 1st step: degradation of uncalcified bone matrix (osteoid); PA0 2nd step :induction of the formation of osteoclasts; and PA0 3rd step: activation of osteoclasts.
In the 1st step, osteoprogenitor cells in marrow are differentiated into osteoblasts and proliferated. In the 2nd step, type I collagen was secreted from activated osteoblasts to form a matrix which is supportive tissues to deposit calcium and phosphorus. Successively, non-collagenous proteins, e.g., osteocalcin and osteonectin etc. are secreted from osteoblasts and are deposited to form bone matrix. In the 3rd step, calcification is carried out by depositing hydroxyapatite crystals [Ca.sub.10 (PO.sub.4).sub.6 (OH).sub.2 ] to the bone matrix.
Osteoblasts have a high alkaline phosphatase activity and contain acidic phospholipids in a high concentration. It is considered that the apatite crystals are produced by their actions.
On the other hand, the bone resorption stage divided into:
The surface of bone is covered with an osteoid consisting of type I collagen as a main component. Osteoid is digestively resorbed by collagenase secreted from osteoblasts (1st step). Migration of osteoclasts to the bone is induced by the action of degradation products of collagenase produced at the former step. Osteoclasts are adhered via adhesion molecules (vitronectin) (2nd step). Carbonate dehydrogenase is produced by the action of activated osteoclasts, and calcium phosphate is dissolved out by the enzyme, and further catepcin L secreted from osteoclasts degradate bone matrix (3rd step).
Interleukin-4 (abbreviated as IL-4 hereafter) is a glycoprotein which T lymphocytes produce when they are stimulated with lectin, phorbol ester or antigen. IL-4 has been identified as a factor in a body, relating to differentiation and proliferation of B lymphocytes or T lymphocytes [see Y. Noma et al., Nature, 319, 640(1986); F. Lee et al., Proc. Natl. Acad. Sci. USA, 83, 2061(1986); E. Severinson et al., Eur. J. Immunol., 17, 67(1987) and T. R. Mosmann et al., Proc. Natl. Acad. Sci. USA, 83, 5654(1986), the disclosures of all of which are incorporated herein by reference].
In 1986, c-DNA of human IL-4 was cloned, thereby revealing that IL-4 is a substance having a molecular weight of 18-21 Kd, consisting of 153 amino acid [see T. Yokota et al., Proc. Natl. Acad. Sci. USA, 83, 5894(1986), the disclosures of all of which are incorporated herein by reference].
In the 153 amino acids, 24 amino acids at the N-terminal form a signal peptide. Therefore, the mature IL-4 has the remaining 129 amino acids (molecular weight of 15 Kd) as a core peptide, attaching sugar chains, and being of a molecular weight of 18-21 Kd as a whole.
According to recent investigation it has been found that IL-4 has biological activities relating to not only B lymphocytes and T lymphocytes but also hematocytes. In particular, it has been found that IL-4 suppressively acts on macrophages and inhibits the release of various kinds of cytokines such as interleukin-1 (IL-1), tumor necrosis factor (TNF), interferon (IFN) etc., derived from macrophages [see P. H. Hart et al., Proc. Natl. Acad. Sci. USA, 86, 3803(1989) and M. Hurme et al., Biochem. Biophys. Res. Commun., 157, 861(1988), the disclosures of all of which are incorporated herein by reference].
More recently, it has been reported that IL-4 inhibited bone resorption stimulated by parathyroid hormone etc. [K. Watanabe et al., Biochem. Biophys. Res. Commun., 172(3), 1035(1990), the disclosures of all of which is incorporated by reference]. It is described therein that the authors conducted their experiment, taking into consideration that IL-4 inhibits the differentiation of hematopoietic stem cells to osteoclasts and the proliferation of osteoclasts and thereby induced the inhibition of bone resorption, because osteoclasts are derived from hematopoietic stem cells as a precursor in common with monocytes/macrophages on which IL-4 may exert effects. The results as expected were given.
On the other hand, it is necessary to provide large amounts of IL-4 for clinical use. Many methods have been developed for transforming a gene of IL-4 into genes of yeast, E. coli, or various mammalian cells and culturing the transformants to produce large amount of the desired IL-4. They are described in detail, for example, in the specifications of the European Patent Publication No. 302429, PCT Publication No. WO 87/02990, and European Patent Publication No. 301835 and 342892, the disclosures of all of which are incorporated by reference herein. Each IL-4 obtained by various methods has fundamentally the same core peptide as natural IL-4 has, in spite of a slight difference, for example, having or not having a sugar chain. Therefore, they are proved to have the same biological activities as natural IL-4 has. Recently, it has been proposed that IL-4 analogues in which a part of the amino acids composing core peptides of human IL-4 be removed or exchanged, or in which other amino acids or other polypeptide be added to core peptides. For example, it is described in the specification of the PCT Publication No. WO 88/04667, the disclosures of all of which is incorporated herein by reference, that IL-4 analogues subjected to the addition or exchange to the core peptides have the same biological activity as natural IL-4 has.